DESCRIPTION: The Specific Aim of this research is to enhance our understanding of the regulation of erythropoiesis, and to apply this information to clinical anemias. Using highly- purified BFU-E and CFU- E, the cellular physiology and biochemistry of the response to erythropoietin, stem cell factor, IL-3 and IGF-1 will be studied. The interaction of these growth-promoting cytokines with inhibitory cytokines, including TNF, IL-1 and the interferons, will also be studied. The basic premise is that critical intracellular signaling molecules are regulated by growth-promoting cytokines, and that the inhibitory cytokines act to either down-regulate the receptors for the former, impact upon the secondary signaling molecules, or impact upon nuclear factors which ultimately lead to growth factor- induced cellular proliferation and differentiation. Experiments are designed to study each of these levels of potential regulation. In the first set of experiments, the investigator plans to determine the specific BFU-E target cell that is maximally inhibited. BFU-E, purified by the investigator, are heterogenous in the amount of time it takes to develop into an observable hematopoietic colony. Previous studies have shown that interferons partially inhibit the growth of BFU-E; in the present study the investigator will determine whether early stage or late stage BFU-E are affected. Once discerned, that population of BFU-E will be used for further studies of the interaction between positive-acting hematopoietic growth factors and negative regulatory cytokines. On the whole colony level, the investigator plans to determine whether SCF, IGF-1, or Epo can overcome the inhibitory effect of cytokines such as interferon gamma. In a second set of studies, the Investigator hypothesizes that inhibitory cytokines affect the number, binding affinity, or internalization of receptors for positively-acting growth factors. As such, in the presence of the interferon gamma, receptors for Epo, IGF-1, and SCF will be determined using standard receptor-binding kinetic studies. Preliminary data suggests that SCF receptors might be downregulated in such a manner. On a second level, the ability of inhibitory cytokines such as interferon gamma to block the tyrosine phosphorylation of the erythropoietin, SCF and IGF-1 receptors, and that of secondary transduction proteins found to be important in signaling of these cytokines will be studied. In addition to the receptors, p91 STAT, JAK-1 and JAK-2 will be studied. Moreover, since phosphorylation is not a functional assay, the Investigator will study the ability of the Epo or SCF stimulated STAT proteins to bind to their cognate DNA site. Finally, the investigator proposes a new specific aim in which he plans to test the effects of interferon gamma and hematopoietic growth factors on the ratio of IRF-1 and IRF-2. These two transcriptional regulators are mutually antagonistic; the ratio of the two has been shown to affect proliferation in a number of cell systems. Dr. Krantz plans to determine the relative effects of interferon gamma and the hematopoietic growth stimulatory cytokines on the ratio of IRF-1 and IRF-2, and to determine the mechanism of alteration of this ratio to determine whether, in a normal erythroid system, these transcriptional factors play an important role.